Year

2005

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences

Abstract

Arsenic methylation is the common pathway of arsenic metabolism in most organisms. Compared with mammalian tissues, information is scant on the enzymes responsible for arsenic methylation in plants and microorganisms. The research in this thesis investigated the arsenic methylation activities in an arsenic-resistant plant species (Agrostis tenuis Sibth) and two common microorganisms: budding yeast (S. cerevisiae) and E. coli.

Plants were grown in complete nutrient media with arsenate (135 to 538 μM) for 3 days before harvesting. Methylation activity was determined in leaf and root tissue extracts using an in vitro assay based on the methyl group transferred from S-[3H-methyl] adenosyl-L-methionine (3H-AdoMet) with either arsenite or arsenate as substrate. Arsenite methylation activity was low in leaf extracts from plants not exposed to arsenate, but was greatly enhanced after acute exposure, with the induced methylation activity greatest in extracts from plants exposed to 269 mM arsenate. Monomethylarsonate (MMA) was the predominant early product, but over longer assay times, dimethylarsinate (DMA) accumulated at the rate of 660 amol.mg protein-1.min-1 to levels exceeding MMA. With arsenate assubstrate, methylation activity was much lower than with arsenite, implying that arsenite is the preferred substrate for methylation. Methylation assays with root extract generated no DMA, however, small amounts of MMA were formed with arsenite as substrate. In contrast to leaves, the methylation activity did not increase in root extracts from plants exposed to arsenate. These findings suggest that arsenate in the plant growth medium was taken up by the roots and converted to arsenite before methylation proceeded in the leaves, accompanied by induction of arsenic methyltransferase activities.

A comparative study was made of the enzymic methylation of arsenic in yeast and E. coli. Research using yeast offers an approach to the information on genes coding for the enzymes responsible for arsenic methylation. Studies investigated the arsenic methylation activities extractable from yeast Saccharomyces cerevisiaeBY4741and its arc3 (arsenite transporter gene) knock-out mutant YPR201w, and from bacterium E. coli W3110 and its ars (arsenic resistance operon) deletion mutant AW3110. These strains were grown in complete media with arsenate for 20 hours (yeast) or 4 hours (E. coli) before harvesting. Methylation activity was determined in cell extracts using the in vitro assay with either arsenite or dimethylarsinate (DMA) as substrate. Arsenite methylation activity was detectable in the extracts from all four strains even when not exposed to arsenate and the activities increased over time. With the BY4741 strain, monomethylarsonate (MMA) was the predominant early product, but over longer assay times, DMA accumulated at the rate of 4.1 amol.mg protein-1.min-1 to levels exceeding MMA. Longer assay times were required for extracts from YPR201w to produce substantial amounts of MMA and DMA, implying that the deletion of acr3 affects arsenic methylation. After acute exposure to arsenate, the methylation activity was highest at 15 minutes with both strains. DMA was produced at the rate of 33.4 amol. mg protein-1.min-1 in BY4741 during a 15 minute assay. The amounts of MMA and DMA in the reaction mixtures decreased after 15 minutes. This phenomenon implied further methylation, which was confirmed by DMA-dependent arsenic methylation assays. The findings suggest that yeast possesses a constitutive arsenic methyltransferase activity, but that exposure to arsenate can stimulate additional higher methylation activity. However, for E. coli wild type W3110, arsenite and MMA methyltransferase activities were not affected by arsenate in the culture medium.

Searching for the gene(s) encoding for arsenic methyltransferases, thirteen genes were chosen from the S. cerevisiae genome database and knockout mutants for each of the 13 genes were obtained. Arsenic methylation by partially purified extracts from these mutants was investigated by the in vitromethylation assay with arsenite as substrate. The yeast knockout mutants were grown in the GYP complete medium without arsenic present. Compared with the other knockout mutants, the mutant lacking functional gene YHR209w showed low rates of formation of MMA and DMA, while the mutant of gene YER175c, which is known to code for a trans-aconitate methyltransferase, formed little MMA but substantial amounts of DMA. Fusion genes of GST-YHR209w and GST-YER175c were highly expressed in yeast stain EJ758. The fusion proteins produced were treated with thrombin to release the target protein from GST. The products of GST-YHR209w with thrombin treatment at 19°C exhibited arsenite methyltransferase activity but were not able to methylate MMA. It is suggested that protein YHR209w is probably responsible for the methyl group transfer to arsenite but not to MMA. This differs from S-adenosyl-L-methionine:arsenic(III) methyltransferase in rat liver cells, which catalyses both arsenite and MMA. Protein YHR209w is an S-adenosyl-methionine-dependent methyltransferase and contains all four typical motifs that have been found in other methyltransferases.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.